冻存保护剂浓度对乳猪肝细胞-80℃低温保存的影响  

Effect of DMSO concentrations on cryopreservation of suckling porcine hepatocytes

作　　者：刘鸿凌[1] 王英杰[1] 郭海涛[1] 刘俊[1] 于乐成[1] 

机构地区：[1]中国人民解放军第三军医大学西南医院全军感染病研究所,重庆市400038

出　　处：《世界华人消化杂志》2002年第12期1392-1395,共4页World Chinese Journal of Digestology

基　　金：全国优秀博士专项基金资助课题NO.199947;国家自然科学专项基金资助项目NO.30027001~~

摘　　要：目的:探索适宜于乳猪肝细胞-80℃长期低温保存的冻存保护剂浓度.方法:体外分离乳猪肝细胞,以含不同浓度二甲基亚砜(DMSO,浓度分别为50、100、150和200mL/L)的低温保存液-80℃分别保存肝细胞30d与60d.复苏后接种培养,对其存活率、贴壁率、白蛋白及尿素合成能力等进行检测,并观察其形态结构变化.结果:不同DMSO浓度的低温保存液保存复苏后猪肝细胞的活力及形态学表现均有所不同,其中含50mL/LDMSO低温保存液组保存效果最差;100mL/L组次之;150mL/L组最佳.150mL/LDMSO低温保存液组保存60d肝细胞复苏后的存活率为81%,贴壁率是79%,较50mL/L与100mL/LDMSO组有显著性差别(P<0.05),150mL/LDMSO组复苏后接种培养肝细胞的形态与未冻存组无明显差别,均保持较强的合成尿素及白蛋白能力.结论:150mL/LDMSO浓度适合于乳猪肝细胞的-80℃长期超低温保存,保存时间对其活力无明显影响.冷冻保存;冻存保护剂;二甲基亚砜.AIM: To determine the best concentration of dimethylsulfoxide (DMSO) for preservation of suckling porcine hepa-tocytes at -80 ℃.METHODS:Primary porcine hepatocytes were harvested bymodified two-step perfusion collagenase method, then storedin the 50, 100, 150 or 200 mL /L DMSO cryopreservation medium.They were cryopreserved for 30 d or 60 d before plated underculture medium conditions. After cold storage, their viability,attachment rate and drug metabolic function were measured,and intracellular structure changes were observed underlight microscope.RESULTS:After storage in 50 mL/L DMSO, cell viability andadherence were significant reduced. But the hepatocytesmaintained in 150 mL/L DMSO solution kept their functionwell, and their albumin secretion, extracellular LDH and drugmetabolic reaction were close to those found in freshhepatocytes.CONCLUSTION:The best concentration of DMSO for –80 ℃cryopreservation of porcine hepatocytes is 150mL/L, in whichprimary porcine hepatocytes can be cryopreserved for 60days.Porcine hepatocytes; Cryopreservation;DMSO;Cryopro-tective agent

关 键 词：冻存保护剂 乳猪 肝细胞 低温保存 生物人工肝 

分 类 号：R657.3[医药卫生—外科学]